TP53RK Drives the Progression of Chronic Kidney Disease by Phosphorylating Birc5

Abstract Renal fibrosis is a common characteristic of various chronic kidney diseases (CKDs) driving the loss of renal function. During this pathological process, persistent injury to renal tubular epithelial cells and activation of fibroblasts chiefly determine the extent of renal fibrosis. In this study, the role of tumor protein 53 regulating kinase (TP53RK) in the pathogenesis of renal fibrosis and its underlying mechanisms is investigated. TP53RK is upregulated in fibrotic human and animal kidneys with a positive correlation to kidney dysfunction and fibrotic markers. Interestingly, specific deletion of TP53RK either in renal tubule or in fibroblasts in mice can mitigate renal fibrosis in CKD models. Mechanistic investigations reveal that TP53RK phosphorylates baculoviral IAP repeat containing 5 (Birc5) and facilitates its nuclear translocation; enhanced Birc5 displays a profibrotic effect possibly via activating PI3K/Akt and MAPK pathways. Moreover, pharmacologically inhibiting TP53RK and Birc5 using fusidic acid (an FDA‐approved antibiotic) and YM‐155(currently in clinical phase 2 trials) respectively both ameliorate kidney fibrosis. These findings demonstrate that activated TP53RK/Birc5 signaling in renal tubular cells and fibroblasts alters cellular phenotypes and drives CKD progression. A genetic or pharmacological blockade of this axis serves as a potential strategy for treating CKDs.


Supplemental Materials
Experimental Section Supplemental Figures

Experimental Section
RNA sequencing (RNA-Seq): UIR and sham operated mice were sacrificed at 1, 3, and 21 days after surgery.Kidney tissues were harvested and frozen rapidly in liquid nitrogen.RNA isolation, library construction, and sequencing were performed by Beijing Genomic Institution using a BGISEQ-500 RNA-seq platform (Beijing Genomic Institution, Shenzhen, China).The mouse GRCm38.p5reference genome was used to map the clean tags.
High-throughput tail vein plasmid delivery: Wild-type C57BL/6J mice (7 weeks old, male) were purchased from GemPharmatech (Nanjing, Jiangsu, China) and were allowed to acclimate to the housing environment for a week.TP53RK overexpression, TP53RK and Birc5 targeted CRISPR/Cas9 plasmids, as well as the corresponding vectors were dissolved in saline to a working concentration of 30 μg/mL before injection.Plasmids amounting to 2 mL were delivered to the tail vein of mice within 10 s.UUO or sham operation was performed 36 h after injection.Animal experiments were performed in the Animal Core Facility of Nanjing Medical University and all of the procedures were approved by the Nanjing Medical University Institutional Animal Care and Use Committee.
Hematoxylin and eosin (H&E) staining: H&E staining was carried out using a commercial kit (Cat #G1120, Solarbio, Wuhan, China) according to the manufacturer's instructions.Briefly, the tissue slides were stained with hematoxylin solution for 10 min, differentiated with differentiation solution for 3 min and re-dyed with eosin Y aqueous solution for 1 min.The tissue slides were viewed and imaged under a Olympus BX51 microscope (Olympus, Tokyo, Japan).

Pharmaceutical combination treatment of UUO mice:
To explore the combinational effect of fusidic acid (FA) and YM-155 treatment in UUO mice, 8 weeks old male C57BL/6J mice (GemPharmatech, Nanjing, China) were randomly divided into five groups: sham group, UUO group, UUO+FA group, UUO+YM-155 group, and UUO+FA+YM-155 group.FA was given to mice at 10 mg/kg/d and YM-155 was employed at 3 mg/kg/d, via intraperitoneal injection 24 h and 2 h before UUO surgery.Then the mice were treated daily for 7 consecutive days and sacrificed 2 h after the final injection.after surgery (n = 3).Expression of genes was measured using fragments per kilobase of transcript per million mapped reads (FPKM).Data are presented as mean ± SEM; two-tailed unpaired t-test was used to determine statistical significance; * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the sham group.were treated with TGF-β1 (10 ng/mL) for 24 h and harvested for western blot analysis of TP53RK, POSTN, and FN.

Figure S10 Pharmaceutical combination treatment of UUO mice with fusidic acid and YM-155
(A-B) FA was given to mice at 10 mg/kg/d and YM-155 was employed at 3 mg/kg/d, via intraperitoneal injection 24 h and 2 h before UUO surgery.Then the mice were treated daily for 7 consecutive days and sacrificed 2 h after the final injection.Deposition of total fibrosis in kidney tissues was determined by Masson's trichrome staining (A).
Scale bar, 20 μm.Quantification analysis of fibrotic area was shown in parallel (B) (n = 6-10).(C) mRNA levels of Vim, Fn, and Col1a1 in kidney tissues of corresponding groups (n = 6-10).(D) Representative immunoblots of VIM and FN in each group.(E) NRK-49Fs were pre-treated with FA (10 μM), YM-155 (2.5 nM) or both of the two inhibitors for 2 h and then stimulated with TGF-β1 (5 ng/mL) for 24 h.Protein expression of α-SMA and FN of each group was estimated.Data are presented as mean ± SEM; one-way ANOVA followed by Dunnett's multiple comparisons test was used to determine statistical significance; *** p < 0.001 compared with the sham group; # p < 0.05, and ### p < 0.001 compared with the UUO group.(H) Mice were pre-treated with YM-155 (3 mg/kg/d) and subjected to sham or UUO operation and euthanized 14 days thereafter.Levels of BUN and sCr in corresponding groups were shown (n = 9-10 in each group).(I) Mice were pre-treated with FA (10 mg/kg/d), YM-155 (3 mg/kg/d) or both of the two agents via intraperitoneal injection 24 h and 2 h before UUO surgery.Then the mice were treated daily for 7 consecutive days and sacrificed 2 h after the final injection.BUN and sCr levels of each group were evaluated (n = 6-10).Data are presented as mean ± SEM; two-way ANOVA followed by Tukey's multiple comparisons test was used to determine statistical significance in

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Figure S1 Expression pattern of TP53RK in fibrotic kidneys

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Figure S2 Genetic or pharmaceutic inhibition of TP53RK alleviated TGF-β1-induced p-EMT in cultured

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Figure S3 Genetic or pharmaceutic inhibition of TP53RK alleviated TGF-β1-induced proliferation and

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Figure S4 Safety assessment of high-throughput tail vein delivery of TP53RK over-expression and

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Figure S5 Global overexpression of TP53RK aggravated UUO-induced kidney fibrosis

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Figure S9 Tubular conditional knockout of TP53RK attenuated AKI-CKD transition

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Figure S11 BUN and sCr levels of UUO and UIR mice

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Figure A-H and the one-way ANOVA followed by Dunnett's multiple comparisons test was used to determine